Translocation of specific polypeptides across membranes is essential for all living cells. The pathway of transfer through the membrane provided by the heterotrimeric complex SecYEG in Escherichia coli is highly conserved in all three kingdoms of life, from single cell organisms to mammals. In addition to a pathway through the membrane in almost all cases, whether the process occurs in prokaryotes or eukaryotes, chaperones are involved in the initial stages of binding of precursor polypeptides that are to be transported. The principles underlying the phenomena of capture and movement of polypeptides through membranes are likely to be shared among different systems. Therefore what we learn by studying bacterial export will provide insights into the molecular mechanisms of other export systems. The research plan is designed to elucidate the mechanistic details of the process with emphasis on the dynamics of passage of a precursor polypeptide along the pathway from the chaperone SecB to SecA, the ATPase motor of the system, and on through the SecYEG translocon. The work will increase the depth of understanding of molecular switches including both changes in specific contacts within complexes that are crucial to the movement of the precursor from one binding partner to another as well as conformation changes within SecA that underlie the conversion of energy of binding and hydrolysis of ATP to the mechanical work of translocation through the channel in the membrane. The research strategy makes use of a balance of biophysical and biochemical techniques that complement one another. The experiments proposed range from determinations of binding parameters by titration calorimetry and of hydrodynamic properties of complexes by light scattering and ultracentrifugation to the complete reconstitution into proteoliposomes of the entire translocation system comprising not only the translocon, but also the accessory proteins SecDF/YajC as well as bacterial rhodopsin as a means to generate protonmotive force. Among the innovative techniques are the use of Nanodiscs that allow membrane proteins such as SecYEG to be treated as a soluble particle and the use of single molecule atomic force microscopy. Many questions of interest cannot be readily answered by ensemble methods because interpretation is confounded by the multiple dynamic equilibria that occur along the export pathway. For these studies assessment of complexes by a single molecule technique is essential. An overall advantage of the research plan is that all methods are carried out in solution conditions and protein concentrations that are as close as possible to physiological. The proposal is firmly based on our past research and should move the field nearer to a molecular description of protein localization.